Alcian Blue Staining Method to Visualize Bonghan Threads Inside Large Caliber Lymphatic Vessels And X-Ray Microtomography to Reveal Their Microchannels

Abstract

Background: Novel threadlike structures, so-called Bonghan threads, inside large caliber lymphatic vessels were recently observed by using the Janus Green B staining in the case of rabbits, and by using fluorescent magnetic nanoparticles for rats. They are thought to have channels through which some liquid with abundant hyaluronic acid (HA) flows. In the present work, the authors put forward a hypothesis to test that the Bonghan threads can be visualized in vivo by HA-staining dye, Alcian blue, and that the channels may be revealed by using X-ray microtomography. Methods and Results: Alcain blue was injected into a lymph vessel near the caudal vena cava of a rat to make the Bonghan threads visible. Specimens were stained with YoYo-1 and Masson's trichrome. They were also examined by confocal microscopy and X-ray microtomography. A Bonghan thread was well stained by Alcian blue and was about 50 μm thick and 10–20 times thinner than the surrounding lymphatic vessel. It had a broken line shape, with a distribution of rod-shaped nuclei, which is the characteristic of Bonghan threads in general. Whereas lymphatic vessels are surrounded by a collagenous matrix, Bonghan threads do not contain any collagenous component. X-ray microtomography revealed continuous microchannels inside the Bonghan threads. Conclusions: Bonghan threads contain HA abundantly, harbor continuous microchannels, and have characteristic distribution of the rod-shape nuclei. Thus, they are novel anatomical structures with liquid-carrying microchannels.